Psoriasis is a skin disorder characterized by inflammatory and abnormal epidermal keratinocyte hyper-proliferation resulting in hyperplasia, thickening of the epidermis and the presence of red scale plaques. The chronic skin condition is recognized for its peculiar clinical symptoms, characterized by circumscribed red patches covered with white scales that result in itchy, flaky skin. Psoriasis is a very visible disease and frequently affects the face, scalp, trunk and limbs. The lesions in this chronic disease typically are subject to remission and excerbations.
Although, psoriasis manifests as a skin disorder, while not being bound to any theory, it is believed to be a disease of impaired or defective cell mediated immunity. Since the clinical appearance of psoriasis is largely caused by epidermal changes, the disease has traditionally been considered one of excessive keratinocyte proliferation and abnormal differentiation. Current evidence suggests that epidermal changes in psoriasis are caused by actions of T lymphocytes in skin lesions and that T lymphocytes induce or sustain the disease process. Psoriasis is portrayed as an autoimmune disease, where activated T-lymphocytes, producing multiple cytokines cause secondary epithelial abnormalities. Dysregulated lymphocytes produce cytokines that stimulate the proliferation of apoptosis-resistant keratinocytes. Psoriatic skin lesions are characterized by inflammation, with T cells and neutrophils infiltrating both the dermis and epidermis and excessive scaling related to epidermal hyperproliferation and aberrant keratinocyte differentiation [Reich K., Garbe C., Blaschke V., Maurer C., Middel P., Westhal G., Lippert U., and Neumann C., J. Invest. Dermatol., 2001, 116, 319].
Autoimmune disorders are diseases caused by the body producing an immune response against its own tissues. The cause of autoimmune diseases is unknown, but it appears that there is an inherited predisposition in many cases in the development of an autoimmune disease.
In a few types of autoimmune disease (such as rheumatic fever), a bacteria or virus triggers an immune response, and the antibodies or T-cells attack normal cells because they have some part of their structure that resembles a part of the structure of the infecting germ.
Autoimmune disorders fall into two general types: those that damage many organs (systemic autoimmune diseases), and those where only a single organ or tissue is directly damaged by the autoimmune process (localized). Some of the most common types of autoimmune disorders are summarized in below:
Systemic AutoimmuneDiseasesLocalized Autoimmune DiseasesRheumatoid arthritis (joints;Type 1 Diabetes Mellitus (pancreas islets)less commonly lung, skin)Lupus [Systemic LupusHashimoto's thyroiditis, Graves' diseaseErythematosus] (skin, joints,(thyroid)kidneys, heart, brain, redblood cells, other)Scleroderma (skin, intestine,Celiac disease, Crohn's disease, Ulcerativeless commonly lung)colitis (GI tract)Sjogren's syndrome (salivaryMultiple sclerosis*, Guillain-Barreglands, tear glands, joints)syndrome (brain)Goodpasture's syndromeAddison's disease (adrenal)(lungs, kidneys)Wegener's granulomatosisPrimary biliary sclerosis, Sclerosing(sinuses, lungs, kidneys)cholangitis, Autoimmune hepatitis (liver)
The inflammatory process involves a series of events that can be elicited by numerous stimuli (e.g. infectious agents, ischemia, antigen-antibody interactions, and thermal or other physical injury). Each type of stimulus provokes a characteristic pattern of response that represents a relatively minor variation on a theme. At a macroscopic level, the response usually is accompanied by the familiar clinical signs of erythema, edema, tenderness and pain.
The symptoms observed in psoriatic patients include hyperplasia and abnormal cornification of epidermal cells ascribed to the excess turnover of the cells by hyper metabolism, asthenia of inflammatory response in the epidermal layer, vasodilation and leukocyte migration and infiltration into the epidermal cell layers. However, it is now recognized that epidermal hyperplasia is a reaction to the activation of immune system in focal skin regions, which in turn, is mediated by CD8+ and CD4+ T lymphocytes that accumulate in the diseased skin. Indeed, psoriasis is now recognized as the most prevalent T cell-mediated inflammatory disease of humans. The symptoms in psoriasis thus appear to be overly rapid growth of keratinocytes and shedding of scales from the skin surface. Within psoriatic lesions, the keratinocyte cell cycle time is reduced approximately 8 fold (36 vs. 311 hours in normal skin) and the number of dividing cell is doubled, resulting in a hyperplastic epidermis. Drug therapy is directed at slowing down this process.
It was found that PUVA therapy depleted lymphocytes in concert with disease improvements. These data are consistent with a role for T cells in pathogenesis. Cyclosporine, a known immunosuppressant was found to have dramatic effects on disease activity. Since cyclosporine has a major inhibitory effect on T cell activation, arguments began to be made that psoriasis was fundamentally an inflammatory disease.
T-lymphocytes must infiltrate the dermis and then adhere to keratinocytes to produce psoriatic plaque. Hence molecular regulating T cell adhesion and trafficking become tenable therapeutic targets and its role in pathophysiology is of considerable importance. Intravascular adhesion events can be inhibited by blocking chemokine triggering or blocking integrin binding (LFA-1 to ICAM-1). Integrin blockade or reduction of its surface expression could be an important event for lymphocytes trafficking which aid in anti-psoriatic therapy.
Immunosuppression, lymphoproliferation inhibition, cytokine modulation such as IL-2 inhibition, IFN-γ inhibition, or IL-10 induction; keratinocyte proliferation inhibition, keratolytic activity and inhibitory activity in MEST are known to be involved in anti-psoriatic activity.
The number of different and sometimes toxic treatments employed for amelioration of psoriasis is testimony to the resistant nature of this disease. As the majority (90%) of psoriasis patients have limited forms of the disease, topical treatments that include dithranol, tar preparations, corticosteroids and the recently introduced vitamin D3 analogues (calcipotriol, calcitriol) can be used. A minority (10%) of psoriasis patients have a more serious condition, for which a number of systemic therapeutic modalities are available. Specific systemic therapies include UVB, PUVA, methotrexate, vitamin A derivatives (acitretin) and immuno-suppressants such as cyclosporin A. The effectiveness of cyclosporin and FK-506 for treating psoriasis provides support for the T cell hypothesis as the prime cause of the disease.
The topical use of corticosteroids reduces the symptoms of psoriasis. However their administration for a long period of time, which is necessary in such treatment causes tachyphylaxis so that either the dose has to be increased or stronger drugs have to be used leading to atrophy and achromasia or loss of pigmentation of peripheral normal skin, when it is topically applied on psoriatic lesion [British National Formulary (BNF), March 2001, No. 41].
Use of phototherapy (irradiation with ultraviolet radiation) or photochemotherapy, which consists of external or internal administration of psoralens and application of long wave ultraviolet rays to the affected part, is associated with disadvantages like the possibility of accelerated aging or pigmentation of the skin and of inducing carcinogenesis [British National Formulary (BNF), March 2001, No. 41].
External use of coal tar, even though is associated with fewer side effects when compared with steroids, is, however, messy and the drawbacks include strong odour, staining of skin etc. Occasionally it may cause stimulant dermatitis.
Methotrexate, even though it is a drug of choice for treating psoriatic conditions, needs to be closely monitored because it can cause liver damage and/or decrease the production of oxygen carrying red blood cells, infection-fighting white blood cells and clot-enhancing platelets. The long-term use of psoralens and methotrexate significantly increase the risk of squamous cell carcinoma in patients with psoriasis [Stern R. S., and Laird N., Cancer, 1994, 73, 2759].
The retinoids such as etretinate are taken internally by patients suffering from intractable psoriasis; however it is teratogenic and likely to accumulate in the body for a longer period of time and hence it is contraindicated in case of pregnancy [Stern R. S., and Laird N., Cancer, 1994, 73, 2759].
Use of macrocyclic immunosuppressive agents such as Cyclosporine, Tacrolimus and Ascomycin may impair kidney function or cause hypertension. Possible side effects of hydroxyurea include anemia and a decrease in white blood cells and platelets.
Calcipotriol, a synthetic vitamin D3 analogue has become one of the widely prescribed treatments for psoriasis. However, it causes significantly more skin irritation than potent topical corticosteroids. The common adverse effects include lesional or perilesional irritation, facial or scalp irritation, or excerbation of psoriasis [Ashcroft D. M., Wan Po A. L., Williams H. C. and Griffiths C. E. M., BMJ, 2000, 320, 963].
Current biotechnology approaches to psoriasis treatment relate to a direct pharmaceutical-mediated attack, either on cell proliferation or on the immune component of the disease. Immunosuppressive immunobiologicals such as Clenoliximab, MEDI-507, ICM3, IDEC-114, SMART Anti-CD3, Zenapax, Amavive, Hul 134, Xanelim, HuMaxCD4, IC747, IDEC-114 IDEC-131, Nuvion, DAB389IL-2, ONTAK and Etarnercept, known to block immune responses at various stages are currently under different phases of clinical trials.
None of the abovementioned treatments are, however, universally safe and effective. The magnitude of the impact of psoriasis is similar to that of other diseases like depression, hypertension and congestive heart failure. The cost of treating the disease averages 800 USD per patient per year in the United States, and the disease can cause significant loss in productivity [Feldman S. R., American Academy of Dermatology, August 2000].
Further, the disease owing to its sporadic course, gives variable response to treatments, which may also have adverse effects. Hence, it is a difficult disease to cure. The devastating nature of psoriasis is emphasized by the extent of the side effects that disease sufferers are willing to endure to attain a remission to a disease that they know will recur sooner or later.
In addition, apart from the clinical manifestations and inconvenience, the psychological impact of the disease on the patient's life is tremendous. Psoriasis is a complex condition affecting all aspects of emotion and physical debilitation for the patient and, substantially reduces the quality of life for millions of people all over the world. Moreover, as it is often clearly visible, affected individuals suffer marked distress, embarrassment and discomfort [Fortune D. G., Richards H. L., Main C. J., and Griffiths C. E. M., J. Am. Acad. Dermatol., 1998, 39, 196].
A composition derived from a plant source, which provides a safe, well-tolerated and effective treatment of psoriasis and which moreover, overcomes the shortcomings and limitations of the current treatments has been disclosed in our US Patent Publication No. 2003/0194456 A1.
US Patent Publication No. 2003/0194456 A1 discloses useful in vitro and in vivo immunological and pharmacological activities of a medication/composition comprising an extract obtained from the leaves and/or stems of the plant, Argemone mexicana, optionally in combination with an extract obtained from the fruits of the plant, Cuminum cyminum for the treatment and prophylaxis of psoriasis and other disorders. The extract, which can be an aqueous, ethanolic or aqueous-ethanolic extract, apart from exhibiting useful immunological and pharmacological activities provides significant reduction in the rate of Psoriasis Area and Severity Index (PASI) score with better tolerability within the range of normal permissible limits. In proof of concept studies conducted on patients having chronic plaque type psoriasis, a composition comprising the abovementioned extract when administered orally was found to result in reduction of the PASI score from 6.33±2.84 to 0.90±1.27, with a disease free state observed in some patients after 8 weeks of treatment.
US Patent Publication No. 2003/0194456 A1 further reports the acute toxicity (LD50) of the extract obtained from the leaves and/or stems of the plant, Argemone mexicana, as evaluated in mice and rats through oral and i.v. routes of administration to be >1000 mg/kg body weight of the animal with 50% mortality.
It might be mentioned herein that the Argemone mexicana plant is composed of various compounds, which include inter alia:
i) Alkaloids such as protopine, protopine nitrate, berberine, berberine nitrate, cryptopine, allocryptopine, coptisine, sanguinarine, dihydrosanguinarine, norsanguinarine, 6-acetonyl dihydrosanguinarine, dihydrochelerythrine, chelerythrine, norchelerythrine, 6-acetonyl dihydrochelerythrine, (−) cheilanthifolin, (−)-β-scoulerine methohydroxide, (−)-α-stylopine (−)-α and β-stylopine methohydroxides, (−)-cheilanthifolin, 6-acetonyl dihydrosanguinarine, (−)-α-tetrahydropalmatine methohydroxide, reticuline, thalifoline, muramine, argemonine, norargeminine, argemexicaine A, argemexicaine B, N-demethyloxysanguinarine; (+)-1,2,3,4-tetrahydro-1-(2-hydroxymethyl-3,4-dimethoxyphenylmethyl)-6,7-methylenedioxy-isoquinoline, helleritrine, and oxyhydrastinine;
ii) Flavonoids, such as isorhmanetin, isorhamnetin-3-glucoside; isorhamnetin-3-O-glucoside, isorhamnetin-3,7-diglucoside; 3-methoxy quercetin, quercetin 5,3′,4′ trimethyl ether; luteolin, argemexitin and eriodictyol;
iii) Fatty acids, such as palmitic, stearic, arachidic, oleic, linoleic, lauric, behenic, lignoceric, hexadecenoic, ricinoleic, 11-oxo-triacontanoic and 11-hydroxy triacontanoic;
iv) Amino acids, such as histidine, lysine, glutamic acid, glycine, alanine, leucine, valine, phenyl alanine, tyrosine, threonine, arginine, serine, asparagine, cysteine, methionine, tryptophan, hydroxyproline, proline, L-glutamine, hydroxyproline, β-alanine, and aspartic acid;
v) Carbohydrates, such as glucose and fructose and glycosides;
vi) Organic acids, such as succinic, citric, tartaric, maleic, and malic; and
(vii) Other compounds like ceryl alcohol, β-sitosterol, potassium nitrate, calcium phosphate and calcium sulphate.
It has been found that the extract obtained from the leaves and/or stem of Argemone mexicana plant using the process for extraction eg. maceration and percolation, using water, ethanol and mixtures thereof disclosed in US Patent Application Publication No. 2003/0194456 A1 contains substantially all of the abovementioned compounds. In other words, the extract is composed of all the compounds present in the parts of the plant used for extraction i.e. it is composed of a mixture of alkaloids, flavonoids, fatty acids, organic acids, amino acids, sugars and salts.
The extracts, thus obtained as per the process described in US Patent Application Publication No. 2003/0194456 A1 were found to exhibit in vitro and in vivo immunological and pharmacological activities eg. immunosuppression, lymphoproliferation inhibition, cytokine modulation such as IL-2 inhibition, IFNγ inhibition, and IL-10 induction; keratinocyte proliferation inhibition, keratolytic activity, endothelial cell proliferation inhibition, inhibition of cell adhesion molecule expression such as ICAM-1, MEST inhibition, and enzymes inhibition such as p60src Tyrosine kinase, which are known to be involved in anti-psoriatic activity.
Furthermore, US Patent Application Publication No. 2003/0194456 A1 teaches that the abovementioned extracts of the leaves and/or stem of Argemone mexicana plant could be fractionated using alcoholic solvents such as n-butanol and methanol and the fractions obtained thereof also exhibit in vitro and in vivo immunological and pharmacological activities including anti-psoriatic activity.
The fractionation procedure of the aqueous extracts of the leaves and/or stem of Argemone mexicana plant, described in Patent Application Publication No. 2003/0194456 A1 was achieved through a multi-step, liquid-liquid partition chromatography, precipitation and drying of extracts and provides fractions containing substantially different classes of compounds as major components.
For instance, as per the method described in US Patent Application Publication No. 2003/0194456 A1 an n-butanol soluble fraction was prepared by adding n-butanol to the aqueous extract of the leaves and/or stem of Argemone mexicana plant, separation of the n-butanol layer from the aqueous phase, followed by washing of the n-butanol layer with water and evaporation of the solvent under reduced pressure to give the n-butanol-soluble fraction as a viscous mass. The aqueous layer, was mixed with methanol, wherein precipitation of a solid mass was effected. The solid mass was separated, and the mass dissolved in water and lyophilized to give the methanol-insoluble fraction. The filtrate obtained after separation of the methanol-insoluble precipitate from the methanol-water mixture on evaporation gave the methanol-soluble fraction as a solid mass.
Typically, Argemone mexicana plant yielded about 3-4.5% of n-butanol-soluble fraction; 46-54% of methanol-soluble fraction, having a total base number between 290-340; and 24-30% of methanol-insoluble fraction, having a total base number between 350-380. As used herein base number is the quantity of acid that is required to neutralize all basic constituents present in 1 g of sample.
As mentioned hereinbefore, the three fractions differ substantially in the constitution of compounds contained therein. The n-butanol-soluble fraction was found to contain alkaloids, flavonoids and other low molecular weight compounds; the methanol-soluble fraction was found to contain amino acids, organic acid and salts; while the methanol-insoluble fraction was found to contain sugars, organic acids and salts.
Even though, extensive chemical investigations over the years on different parts of the Argemone mexicana plant have resulted in the isolation of a number of alkaloids, flavonoids, amino acids, organic acids, fatty acids etc., however, no systematic study has been conducted and reported for isolation and identification of other principles present in the plant.
One such principle, commonly found in the plant kingdom is Arabinogalactan-Proteins (AGP), which are essentially macromolecules of polysaccharides in which the carbohydrate is associated with or linked to proteins. AGP is composed mainly of arabinose and galactose residues. These occur in plants as polysaccharides in association with varying amounts of proteins, and generally contain a high proportion of carbohydrates with comparatively less proportion of proteins, usually less than 10 of proteins, although, AGPs having higher contents of proteins are also known. AGPs are widely distributed in most of the higher plants such as Echinacea purpurea, Nicotiana alata, Vitis vinifera, Diospyros kaki, Gladiolus gandavensis, Lolium multiflorum [Anderson, R. L., Clarke, A. E., Jermyn, M. A., Knox, R. B. and Stone, B. A., Australian J. Plant Physiol., 1977, 4, 143-158], Phaseolus vulgaris [Hawes, G. B., Adams, G. A., Phytochemistry, 1972, 11, 1461-1465] and Acacia arabica [Classen, B., Witthohn, K., and Blaschek, W., Carbohydrate Research, 2000, 327, 497-504].
AGPs possess adhesive and water holding properties and respond to wounds and infections in plants. These determine cellular identity and specific interactions. They also play a role in cell and tissue differentiation as well as in controlling somatic embryogenesis. They are also valued for various biological activities [WO 01/00682 A1, 2001]. There are two types of AGPs, viz. AGP I and AGP II. The latter i.e. AGP II contain a galactose core and are highly branched, contain usually less than 10% proteins and possess side chains highly substituted by arabinofuranosyl residues and sometimes other sugars like rhamnose, glucose, mannose etc. Presence of uronic acids and substituted derivatives are also reported.
As mentioned hereinbefore, even though, chemical investigations on all parts of Argemone mexicana have been conducted, however, no study has been directed towards isolation, characterization and understanding of the biological properties of AGPs present in certain parts of the plant.